title: Optimizing the purification and analysis of miRNAs from urinary exosomes.
abstract: BACKGROUND
  Exosomes are cytoplasm containing vesicles released by many cells that can be found in several biological fluids including urine. Urinary exosomes are released from every segment of the nephron, are detectable in urine, constitutively contain RNA (small RNAs and mRNAs) and harbor unique subset of proteins, reflecting their cellular source.
  
  
  METHODS
  With the aim of establishing the optimal protocol for high throughput analysis of exosomal miRNAs, we compared three different urinary exosomes isolation methods and six RNA extraction techniques. Exosomal RNA yield, size and quality were assessed respectively by specific staining with fluorescent dye, capillary electrophoresis and analysis of spectrophotometric parameters. MiRNAs detection and abundance was determined by RT-qPCR.
  
  
  RESULTS
  Among the exosomes isolation methods, Ultrafiltration resulted to be the most suited. The highest exosomal RNA yield quantified by RiboGreen® staining was obtained with the combination of TRI Reagent™ with miRNeasy®, followed by TRI Reagent™, SeraMir™, miRCURY™, mirVana™ and miRNeasy®; but after a multivariate analysis, SeraMir™ scored as the method of choice in terms of miRNA yield, purity and RT-qPCR miRNAs quantification accuracy. Storage conditions were also analyzed, showing that the relative abundance of urinary exosomal miRNAs is not influenced by urine freezing.
  
  
  CONCLUSIONS
  The selection of appropriate urinary exosomal miRNA isolation method was dependent on various validation results. Ultrafiltration in combination with SeraMir™ exoRNA columns represents the optimal procedure for a rapid, cost-effective and efficient purification of miRNAs from urinary exosomes, perfectly suited for further applicative research in the field of miRNAs in kidney physiology and pathology.
authors.forename: Sarath Kiran
authors.initials: SK
authors.lastname: Channavajjhala
authors.forename: Marzia
authors.initials: M
authors.lastname: Rossato
authors.forename: Francesca
authors.initials: F
authors.lastname: Morandini
authors.forename: Annalisa
authors.initials: A
authors.lastname: Castagna
authors.forename: Francesca
authors.initials: F
authors.lastname: Pizzolo
authors.forename: Flavia
authors.initials: F
authors.lastname: Bazzoni
authors.forename: Oliviero
authors.initials: O
authors.lastname: Olivieri
mesh_terms: D001483:Base Sequence; D005591:Chemical Fractionation / Q000379:methods*; D055354:Exosomes / Q000235:genetics*; D006801:Humans; D007668:Kidney / Q000166:cytology*; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification*; D013997:Time Factors; D016482:Urinalysis / Q000379:methods*
chemical_list: D035683:MicroRNAs